Haynes Lab:Notebook/David Barclay Undergrad Training/Plasmid Transformation/Entry Base: Difference between revisions
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Aims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration | Aims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration. | ||
Protocol: <br> | Protocol: <br> | ||
[[Image:Lab01.jpg|200px|]] | Using Qiaga Protocol,shown below. <br> | ||
[[Image:Lab01.jpg|200px|]] <<br> | |||
Miniprep followed exactly <br> | |||
=====Pre-Miniprep===== | |||
1. Add 3 mL LB into 15 mL tube <br> | |||
2. Add one colony of KAH06 into 3mL LB Broth: Incubate 7 hrs at 37 C. | |||
=====Miniprep===== | |||
1. Pipette 1.5 mL of sample into 2 mL tube. Spin for 3 min @ max setting (13.1g). Repeat once more to spin entire sample. <br> | |||
NOTE: REMEMBER TO MARK TUBES <br> | |||
2. Take 600 μL of sample. Add 100 μL 7x lysis buffer (blue) and invert 4 times. Time limit 2 mins for lysis. <br> | |||
3. Add 350 μL Neutralization Buffer (yellow) invert until sample become yellow (10 times). Neutralization Buffer must be refrigerated. <br> | |||
4. Centrifuge 2 mins @ 13.1g. <br> | |||
5. Transfer ~850 μL into spin column. Put spin column into collection tube. Centrifuge 15 secs @ 13.1g. Discard flow through. <br> | |||
7. Add 200 μL of elution buffer (ENDO Wash Buffer). Centrifuge 15 secs @ 13.1g. <br> | |||
8. Add 400 μL of Zyppy wash buffer into column. Centrifuge 30 secs @ 13.1g. <br> | |||
9. Transfer column into new 1.5 mL tube. Add 30 μL of Zyppy Buffer and let sit for 1 min. Centrifuge 15 secs @ 13.1 g. <br> | |||
=====DNA Concentration- Take 3 Plate===== | |||
1. Open program Gen 5 2.0 before machine. <br> | |||
2. Add 2 μL of sample into holes. <br> | |||
3. Use either water or buffer as blank depending on buffer DNA was added to. <br> | |||
4. Open program "Nucleic Acid Quantification" <br> | |||
5. Use DI water and kim wipe to clean plate. <br> | |||
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Revision as of 12:44, 30 June 2013
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
Tuesday, June 25thSteps for TransformationUsing KAH06 for Plasmid.
Friday, June 28thMiniprepAims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration. Protocol: Pre-Miniprep1. Add 3 mL LB into 15 mL tube Miniprep1. Pipette 1.5 mL of sample into 2 mL tube. Spin for 3 min @ max setting (13.1g). Repeat once more to spin entire sample. DNA Concentration- Take 3 Plate1. Open program Gen 5 2.0 before machine.
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